Namely the WWTP effluent and four points in the river: one point upstream and three points downstream of the discharge of the WWTP. In a more comprehensive experiment, zebrafish liver cells were exposed 48-h in the laboratory to water samples collected along six critical points of the urban water cycle ( Zhen et al., 2018). The impact of a WWTP effluent was assessed through a metabolomics approach by Huang et al. The crustacean was exposed to two stages of wastewater, the prechlorinated wastewater and the final effluent, the latest also with a concentration gradient of added PFOS. (2019) studied different stages of wastewater treatment in lab exposure experiments with D. (2018) in their study with fathead minnows, which were exposed to control water or treated wastewater effluent at 5%, 20%, and 100% levels for 21 days, using an on-site, flow-through system. Similar experimental setup was used by Mosley et al. (2017) exposed rainbow trout and roach fish, respectively, to both WWTP-treated effluent and a clean water sample in a laboratory mesocosms. In addition, experimental setup always includes not only the group of organism exposed to contaminated environment but also a reference or control group of organisms in order to compare xenometabolome and/or metabolome alterations between the two groups. These exposure experiments under controlled conditions allow maintaining all the organisms under the same general conditions: air and water temperature, pH, hours of light/dark, and oxygen ( David et al., 2017). In the case of studies carried out at laboratory scale (mesocosms facilities), a bunch of the studies have been performed using real contaminated water ( Table 10.1). These types of studies with real contaminated water can be performed both in laboratory or directly in the field, where organisms are “caged” for the time of exposure and further processed in the laboratory for the metabolomics and/or xenometabolomics studies. Diana Álvarez-Muñoz, in Environmental Metabolomics, 2020 2.1.2 Experiments performed using real contaminated waters
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